Methods and systems for optimizing perfusion cell culture system

Abstract

Methods and perfusion culture systems are disclosed. The systems and methods relate to decreasing the starting perfusion rate, resulting in increased residence time of the cells in the bioreactor and the cell retention device, and/or concomitantly increasing the starting bioreactor volume or decreasing the starting cell retention device volume, or both. Other method embodiments include increasing the concentrations of individual components of the tissue culture fluid, and adding a stabilizer of the degradation of the recombinant protein.

We claim: 
     
         1 . A perfusion bioreactor culture system, comprising:
 a bioreactor configured to contain a tissue culture fluid and cells to be cultured;   a cell retention device configured to receive tissue culture fluid containing cells from the bioreactor, separate some fluid from the tissue culture fluid and provide harvest output of tissue culture fluid and cells, and provide a recirculation output of tissue culture fluid and cells to the bioreactor;   wherein the system has a starting perfusion rate, a starting bioreactor volume, a starting cell retention device volume, and a starting volume ratio of the starting bioreactor volume and the starting cell retention device volume;   wherein either   the starting perfusion rate is decreased, resulting in increased residence time of the cells in the bioreactor and the cell retention device, or   the starting bioreactor volume is increased or the starting cell retention volume is decreased, or both, resulting in an increase in the starting volume ratio.   
     
     
         2 . The perfusion bioreactor culture system of  claim 1 , wherein the starting perfusion rate is decreased, resulting in increased residence time of the cells in the bioreactor and the cell retention device, and the starting bioreactor volume is increased or the starting cell retention volume is decreased, or both, resulting in an increase in the starting volume ratio. 
     
     
         3 . The perfusion bioreactor culture system of  claim 2 , wherein the increase in the starting volume ratio is about the same proportion as the decrease in the starting perfusion rate. 
     
     
         4 . The perfusion bioreactor culture system of  claim 2 , wherein the starting perfusion rate is decreased by up to about a third. 
     
     
         5 . The perfusion bioreactor culture system of  claim 2 , wherein the starting perfusion rate is decreased by up to about a half. 
     
     
         6 . The perfusion bioreactor culture system of  claim 2 , wherein the starting bioreactor volume is increased by about a third. 
     
     
         7 . The perfusion bioreactor culture system of  claim 2 , wherein the starting bioreactor volume is increased by up to about a half. 
     
     
         8 . The perfusion bioreactor culture system of  claim 2 , wherein the starting cell retention volume is decreased by up to about a third. 
     
     
         9 . The perfusion bioreactor culture system of  claim 2 , wherein the starting cell retention volume is decreased by up to about a half. 
     
     
         10 . The perfusion bioreactor culture system of  claim 2 , wherein the cells are mammalian cells. 
     
     
         11 . The perfusion bioreactor culture system of  claim 10 , wherein the mammalian cells are selected from the group consisting of BHK cells, CHO cells, HKB cells, HEK cells, and NS0 cells. 
     
     
         12 . The perfusion bioreactor culture system of  claim 11 , wherein the mammalian cells are BHK cells. 
     
     
         13 . The perfusion bioreactor culture system of  claim 10 , wherein the mammalian cells are recombinant cells expressing recombinant factor VIII (rhFVIII). 
     
     
         14 . The perfusion bioreactor culture system of  claim 13 , wherein the rHFVIII is an active ingredient of KG-FS. 
     
     
         15 . The perfusion bioreactor culture system of  claim 2 , wherein the starting perfusion rate is about 1 to 15 volumes per day. 
     
     
         16 . The perfusion bioreactor culture system of  claim 2 , wherein the increase in the starting volume ratio is up to about a third. 
     
     
         17 . The perfusion bioreactor culture system of  claim 2 , wherein the increase in the starting volume ratio is up to about a half. 
     
     
         18 . A method of optimizing a perfusion bioreactor system, comprising:
 providing tissue culture fluid containing cells to a bioreactor system comprising a bioreactor and a cell retention device, wherein the system has a starting perfusion rate, a starting bioreactor volume, a starting cell retention device volume, and a starting volume ratio of the starting bioreactor volume and the starting cell retention volume; and   either decreasing the starting perfusion rate, resulting in increased residence time of the cells in the bioreactor and the cell retention device, or   increasing the starting bioreactor volume or decreasing the starting cell retention device volume, or both, resulting in an increase in the starting volume ratio.   
     
     
         19 . The method of  claim 18 , further comprising:
 decreasing the starting perfusion rate, resulting in increased residence time of the cells in the bioreactor and the cell retention device, and   increasing the starting bioreactor volume or decreasing the starting cell retention device volume, or both, resulting in an increase in the starting volume ratio.   
     
     
         20 . The method of  claim 18 , wherein the increase in the starting volume ratio is in about a same proportion as the decrease in the starting perfusion rate. 
     
     
         21 . The method of  claim 18 , wherein the starting perfusion rate is decreased by up to about a third. 
     
     
         22 . The method of  claim 18 , wherein the starting perfusion rate is decreased by up to about a half. 
     
     
         23 . The method of  claim 18 , wherein the starting bioreactor volume is increased by up to about a third. 
     
     
         24 . The method of  claim 18 , wherein the starting bioreactor volume is increased by up to about half. 
     
     
         25 . The method of  claim 18 , wherein the starting cell retention volume is decreased by up to about a third. 
     
     
         26 . The method of  claim 18 , wherein the starting cell retention volume is decreased by up to about a half. 
     
     
         27 . The method of  claim 18 , wherein the cells are mammalian cells. 
     
     
         28 . The method of  claim 27 , wherein the mammalian cells are selected from the group consisting of BHK cells, CHO cells, HKB cells, HEK cells, and NS0 cells. 
     
     
         29 . The method of  claim 27 , wherein the mammalian cells are BHK cells. 
     
     
         30 . The method of  claim 26 , wherein the mammalian cells are recombinant cells expressing recombinant human factor VIII (rhFVIII). 
     
     
         31 . The method of  claim 29 , wherein the rHFVIII is an active ingredient of KG-FS. 
     
     
         32 . The method of  claim 18 , wherein the starting perfusion rate is about 1 to 15 volumes per day. 
     
     
         33 . The method of  claim 18 , wherein the increase in the starting volume ratio is up to about a third. 
     
     
         34 . The method of  claim 18 , wherein the increase in the starting volume ratio is up to about a half. 
     
     
         35 . The method of optimizing a perfusion bioreactor system, comprising:
 providing a first tissue culture fluid containing cells to a bioreactor system comprising a bioreactor and a cell retention device, wherein the system has a starting perfusion rate, a starting bioreactor volume, and a starting cell retention volume; and   decreasing the starting perfusion rate, resulting in increased residence time of the cells in the bioreactor and the cell retention device, and substituting the first tissue culture fluid for a second tissue culture fluid that has, compared to the first tissue culture fluid, increased concentrations of individual components of the first tissue culture fluid, without adding new components.   
     
     
         36 . The method of  claim 35 , wherein the cells are mammalian cells. 
     
     
         37 . The method of  claim 35 , wherein she mammalian cells are selected from the group consisting of BHK cells, CHO cells, HKB cells, HEK cells, and NS0 cells. 
     
     
         38 . The method of  claim 36 , wherein the mammalian cells are BHK cells. 
     
     
         39 . The method of  claim 35 , wherein the mammalian cells are recombinant cells expressing recombinant human factor VIII (rhFVIII). 
     
     
         40 . The method of  claim 39 , wherein the rHFVIII is as active ingredient of KG-FS. 
     
     
         41 . The method of optimizing a perfusion bioreactor system, comprising:
 providing a first tissue culture fluid containing cells that express a recombinant protein to a bioreactor system comprising a bioreactor and a cell retention device, wherein the system has a starting perfusion rate, a starting bioreactor volume, and a starting cell retention device volume; and   decreasing the starting perfusion rate, resulting in increased residence time of the cells in the bioreactor and the cell retention device, and adding a stabilizer of the degradation of the recombinant protein.   
     
     
         42 . The method of  claim 41 , wherein the cells are mammalian cells. 
     
     
         43 . The method of  claim 42 , wherein the mammalian cells are selected from the group consisting of BHK cells, CHO cells, HKB cells, HEK cells, and NS0 cells. 
     
     
         44 . The method of  claim 42 , wherein the mammalian cells are BHK cells. 
     
     
         45 . The method of  claim 42 , wherein the mammalian cells are recombinant cells expressing factor VIII (rhFVIII). 
     
     
         46 . The method of  claim 45 , wherein the rHFVIII is an active ingredient of KG-FS. 
     
     
         47 . The method of  claim 41 , wherein the stabilizer is calcium.